Diagnostic peptides of human papilloma virus

ABSTRACT

A series of seventeen synthetic peptides which are capable of raising antibodies specific for certain desired human papilloma virus (HPV) are useful in diagnosis and therapy of conditions associated with HPV infection.

This is a continuation of U.S. Ser. No. 221,276 filed on Jul. 19,1988,and now abandoned which is in turn a division of U.S. Ser. No.884,184, filed on Jul. 10, 1986, now issued as U.S. Pat. No. 4,777,239.

TECHNICAL FIELD

The invention relates to vaccines and diagnostics relevant to humanpapilloma virus (HPV) infection. In particular, synthetic peptidescorresponding to regions of putative peptides for types of HPV whichinfect the genital region raise antibodies useful in diagnosis and inprotection against infection.

BACKGROUND ART

Human papilloma virus appears to be associated with the development ofcervical carcinoma, a malignant condition which appears to be precededby several stages of cervical intraepithelial neoplasia (CIN). Theassociation of HPV infection with CIN has long been recognized (Meisels,A., et al, Gynecol Oncol (1981) 12:5111-5123; Crum, C. P., et al, ibid(1983) 15:88-94; Syrjanen, K. J., Obstet Gynecol Surv (1984)39:252-265). In fact, IgG reactive with a group-specific papilloma virusantigen was detected in 93% of women with cervical carcinoma and 60% ofthose with CIN, but not in any control subjects (Baird, P. J., Lancet(1983) 2:17-18), and the presence of HPV DNA in these lesions has beenrecognized by several groups.

There are approximately forty different types of HPV, which areclassified by DNA sequence homology using hybridization techniques.Samples having more than 50% homology, as judged by hybridization, areplaced into the same type designation. The various types appear to berather tissue specific. HPV-6, HPV-11, HPV-16, HPV-18, and HPV-31 appearto be associated with the genital tract; others appear to be associatedwith warts or epidermal dysplasias in other tissues. However, HPV-6 andHPV-11 are associated with condyloma type lesions, while HPV-16, HPV-18and HPV-31 are associated with cervical intraepithelial neoplasia,including invasive carcinoma.

The relationship of HPV infection to the development of CIN and cervicalcarcinoma is unclear, however it has been postulated that HPV acts as aninitiator in cervical carcinogenesis and that malignant transformationdepends on interaction with other factors (Zur Hausen, H., et al, Lancet(1982) ii:1370). The incidence of HPV infection appears to be increasingas shown by a 700% increase in patient visits related to genital HPVinfections in both males and females between 1966 and 1981 (Center forDisease Control: Nonreported Sexually Transmitted Diseases (1979) MMWR28:61) and the presence of HPV in more than 3% of pap smears of womenunder 30 (Ferenczy, A.. et al, Am J Surg Pathol (1981) 5:661-670).

The nature of HPV-16 in particular, and papilloma viruses in general hasbeen well studied recently. HPV-16 is a member of the Papova virus groupand contains a 7904 bp double-stranded DNA genome (Siedorf, K., et al,Virology (1985) 145:181-185). The capsid is 50 nm and contains 72capsomers (Klug, A., J Mol Biol (1965) 11:403-423). There are a numberof subtypes of HPV-16 which are isolates showing greater than 50%homology (Coggin, Cancer Research (1979) 39:545-546), but differences inrestriction in endonuclease patterns.

The DNAs of several papilloma viruses have been sequenced, includingseveral HPV types, bovine papilloma virus (BPV) and cottontail rabbitpapilloma virus (CRPV). All of these display similar patterns ofnucleotide sequence with respect to open reading frames. The openreading frames can be functionally divided into early region (E) andlate regions (L); the E region is postulated to encode proteins neededfor replication and transformation: and L region to encode the vitalcapsid proteins (Danos, O., et al, J Invest Derm (1984) 83:7s-11s).

The detection of HPV in cervical samples has been difficult becausethere is no tissue culture system capable of supporting virus harvestedfrom the tissue to be tested through its replication cycle (Tichman, etal, J Invest Derm (1984) 83:25-65). There is, however, a recentlyreported in vitro transformation assay (Yasumoto, S. J Virol (1986) 57:572-577), Tsurokawa, U. et al Proc Nat'l Acad Sci (USA) (1986) 83:2200-2203. It is believed that because of analogy with the betterstudied BPV and CRPV systems, the proteins encoded by several early openreading frames, for example, E6, E5, E7 and E2 (see FIG. 1) areimportant in HPV genital infections. However, no system for utilizationof peptides associated with these regions has been suggested either asan aid to diagnosis or in the synthesis of a vaccine.

DISCLOSURE OF THE INVENTION

The present invention provides peptides selected on the basis ofpredicted secondary structure and hydrophilicity from proteins orpeptides encoded by selected open reading frames. The secondarystructure and hydrophilicity are deduced from the amino acid sequence ofthese proteins according to methods disclosed by Hopp, T., et al, ProcNatl Acad Sci (USA) (1981) 78:3824; Levitt, M., J Mol Biol (1976)104:59; and Chou, P., et al, Biochem (1974) 13:211. The results of thesedeductions permit the construction of peptides which elicit antibodiesreactive with the entire protein, as is further described below. Twogeneral types of such antigenic peptides are prepared. Peptide regionsidentified as being specific to HPV-16 or other HPV type-specificdeterminants by lack of homology with other HPV types lead to thepeptides which are useful to raise antibodies for diagnostic,protective, and therapeutic purposes against HPV-16 or other virus typeper se. Peptide regions which are homologous among the various types ofHPV of interest are useful as broad spectrum diagnostics and vaccines,and elicit antibodies that are broad spectrum diagnostics.

Accordingly, in one aspect, the invention is directed to peptides of thefollowing sequences deduced from the noted regions of HPV-16:

(1) Ser-Arg-Ser-Ser-Arg-Thr-Arg-Arg-Glu-Thr-Gln-Leu (SEQ ID NO:1)(representing residues 147-158 of E6 except Ser was substituted for Cysat position 1):

(2) Phe-Gln-Asp-Pro-Gln-Glu-Arg-Pro-Arg-Lys-Leu- Pro-Gln-Leu-Cys (SEQ IDNO:2), representing residues 9-23 of E6;

(3) Thr-Glu-Leu-Gln-Thr-Thr-Ile-His-Asp-Ile-Ile-Leu-Glu-Cys (SEQ IDNO:3), representing residues 24-37 of E6;

(4) Leu-Arg-Arg-Glu-Val-Tyr-Asp-Phe-Ala-Phe-Arg-Asp-Leu-Cys (SEQ IDNO:4), representing residues 45-58 of E6;

(5) Asp-Lys-Lys-Gln-Arg-Phe-His-Asn-Ile-Arg (SEQ ID NO:5), representingresidues 127-136;

(6) Gly-Pro-Ala-Gly-Gln-Ala-Glu-Pro-Asp-Arg-Ala (SEQ ID NO:6),representing residues 40-50 of E7;

(7) Asp-Thr-Pro-Thr-Leu-His-Glu-Tyr-Met (SEQ ID NO:7), representingresidues 4-12 of E7;

(8) Asn-Asp-Ser-Ser-Glu-Glu-Glu-Asp-Glu-Ile-Asp-Gly (SEQ ID NO:8),representing residues 29-40 of E7;

(9) Leu-Gln-Leu-Thr-Leu-Glu-Thr-Ile-Tyr-Asn-Ser (SEQ ID NO:9),representing residues 75-85 of E2;

(10) Ile-Ile-Arg-Gln-His-Leu-Ala-Asn-His-Pro (SEQ ID NO:10),representing residues 210-219 of E2;

(11) His-Pro-Ala-Ala-Thr-His-Thr-Lys-Ala-Val-Ala-Leu-Gly (SEQ ID NO:11),representing residues 218-230 of E2;

(12) Ser-Glu-Trp-Gln-Arg-Asp-Gln-Phe-Leu-Ser-Gln-Val (SEQ ID NO:12),representing residues 339-350 of E2;

(13) Asp-Gln-Asp-Gln-Ser-Gln-Thr-Pro-Glu-Thr-Pro (SEQ ID NO:13),representing residues 48-58 of E4;

(14) Gly-Ser-Thr-Trp-Pro-Thr-Thr-Pro-Pro-Arg-Pro-Ile-Pro-Lys-Pro (SEQ IDNO:14), representing amino acids 20-34 of E4;

(15) Arg-Leu-Tyr-Leu-His-Glu-Asp-Glu-Asp-Lys-Glu-Asn (SEQ ID NO:15),representing amino acids 476-487 of E1; and

(16) Ala-Pro-Ile-Leu-Thr-Ala-Phe-Asn-Ser-Ser-His-Lys-Gly-Cys (SEQ IDNO:16), representing amino acids 218-230 of E2;

wherein all the foregoing are derived from Type 16; and

(17) Glu-Ser-Ala-Asn-Ala-Ser-Thr-Ser-Ala-Thr-Thr-Ile-Cys (SEQ ID NO:17),representing amino acids 6-17 of the E6 reading frame of Type 6B.

Each of the foregoing peptides, designated herein peptide 1, peptide 2,etc, i.e. peptides 1-17, may, if it does not already have this residue,be prepared with an additional C-terminal cysteine for ease inconjugation to a neutral carrier, and in another aspect, the inventionrelates to peptides containing this additional cysteine.

In addition, the invention relates to a method for synthesizing peptidesuseful in the various aspects of the invention, which method comprisespreparing an analysis of the secondary structure and hydrophilicity ofpeptides encoded by the open reading frames of the DNA corresponding toHPV vital types associated with genital infection, and selecting regionsof secondary structure corresponding to areas on the surface capable ofeliciting antibodies reactive with the entire protein. The selectedpeptide regions are then prepared synthetically using either solid phasesynthesis or other suitable techniques. The invention also relates topeptides prepared using this method. The peptides thus prepared can bedesigned as specific to a particular virus type or may be capable ofraising antibodies cross-reacting against the range of HPV associatedwith genital infection depending on the use desired. Like the specificpeptides numbered 1-17 above, these may also be prepared with aC-terminal cysteine for ease in conjugation.

In further aspects, the invention relates to the foregoing peptidesconjugated to carriers capable of conferring immunogenicity on thesepeptides, to antisera raised against these peptides and the antibodiescontained in these sera, to methods of diagnosing the presence of HPV intissue utilizing these antisera, to methods of detecting anti-HPVantibodies using the peptides per se, and to kits useful in such assays.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the distribution of open reading frames in variouspapilloma viruses.

FIG. 2 shows comparison of the amino acid sequences in the E2 openreading frame for various human papilloma viruses.

FIG. 3 shows comparison of the amino acid sequences in the E6 openreading frame for various human papilloma viruses.

FIG. 4 shows comparison of the amino acid sequences in the E7 openreading frame for various human papilloma viruses.

FIG. 5 shows comparison of the amino acid sequences in the L1 openreading frame for various human papilloma viruses.

FIG. 6 shows comparison of the amino acid sequences in the L2 openreading frame for various human papilloma viruses.

FIG. 7 shows a typical-analysis plot using the referenced methods toascertain secondary structure in hydrophilicity.

FIG. 8 shows the staining of a cervical biopsy at stage CIN2 usingimmunoperoxidase staining mediated by the antibodies of the invention:

FIG. 8A shows the section stained with immune serum preabsorbed with theimmunizing peptide, FIG. 8B shows the same section stained with theimmune serum, and FIG. 8C shows a magnification of the stain in FIG. 8B.

FIG. 9 shows the ability of the immunoperoxidase stain mediated byantibody of the invention to detect a single malignant cell.

MODES OF CARRYING OUT THE INVENTION

The peptides of the invention are used to raise antibodies, either insubjects for which protection against infection by HPV is desired, i.e.as vaccines or to heighten the immune response to an HPV infectionalready present. They are also injected into production species toobtain antisera useful in diagnosis. In lieu of the polyclonal antiseraobtained in the production subjects, monoclonal antibodies may beproduced using the method of Kohler and Milstein or by more recentmodifications thereof by immortalizing spleen or otherantibody-producing cells from injected animals to obtainantibody-producing clones.

In any event, the polyclonal or monoclonal antibodies obtained areuseful for diagnosis of HPV infection in cervical biopsies or pap smearsand in assessing disease levels in human or other subjects. Inparticular, diagnosis using the antibodies of the invention permitsidentification of patients at high risk for malignant transformation aswell as identification of the particular phase of CIN associated withthe sample. The antibodies can also be used in analysis of serum todetect the virus or to detect the virus in metastases of infectedtissue, as well as to monitor the progress-of therapy with antiviral orother therapeutic agents directed to control of the infection orcarcinoma. The antibodies, if corrected for species variations can alsobe used as passive therapy.

In a converse diagnosis, the peptides of the invention can be used insimilar immunoassays to detect the presence of antibodies raised againstHPV in the serum of persons suspected of harboring HPV infections or totitrate the sera of persons treated with an anti-HPV vaccine. The use ofsynthetic peptides for this purpose has the advantage of providing arelatively low cost and reproducible reagent for use in these tests.

Direct administration of the proteins to a host can confer eitherprotective immunity against HPV or, if the subject is already infected,a boost to the subject's own immune response to permit more effectivecombat of the progress of the disease. For all applications, thepeptides are administered in immunogenic form. Since the peptides arerelatively short, this necessitates conjugation with an immunogenicityconferring carrier material. This carrier material should ideally beantigenically neutral--i.e., ineffective in raising antibodies againstitself. Antigenic neutrality is, of course, an ideal state as manycarriers which are actually satisfactory do contain some antigenicregions which are capable of raising antibodies in the host. However,this may still be acceptable if the antigenic regions are in factdifferent from those of the peptide of interest, which is quite easy toachieve, or if the antibodies raised against the carrier portions areharmless to the subject.

The peptides of the invention are designed for their in vitro synthesisby choosing appropriate regions of the protein encoded by the openreading frames of the HPV types of interest. Regions are chosen fortheir immunogenic capability and, depending on the use required, fortheir ability to serve as type specific or broad range vaccines anddiagnostics.

Once designed, the peptides of the invention are prepared by anyconvenient means, commonly by chemical peptide synthesis using solidphase techniques. For conjugation to carrier protein, it is convenientto synthesize these peptides with an additional cysteine residue, forexample, at the C-terminus to provide a sulfhydryl group for convenientlinkage. Depending on the nature of the linkers used, however, otherapproaches to form the conjugates are possible. The conjugated peptidesare then administered to subject animals.

If the peptides are to be administered as vaccines, they are formulatedaccording to conventional methods for such administration to the subjectto be protected. If they are to be used directly, as diagnosticreagents, they are purified and packaged for such use. If they are to beused to produce antibodies for diagnostic purposes, convenient testanimals can be used to prepare the appropriate antisera, and theseantisera used directly. Suitable hosts include mice, rats, rabbits,guinea pigs, or even larger mammals such as sheep. For administration tosuch animals, the peptides linked to carrier are generally administeredin the presence of an adjuvant, usually Freund's complete adjuvant, andthe polyclonal sera are harvested periodically by standard techniques.

If the antibodies are to be used for therapeutic purposes, it isgenerally desirable to confer species characteristics upon themcompatible with the subject to be treated. Accordingly, it is oftendesirable to prepare these antibodies in monoclonal form since fusionwith suitable partners is capable of conferring the desiredcharacteristics on the secreted monoclonals.

These matters are set forth in greater detail below.

Selection of Peptides

At least five HPV types are known to be associated with genitalinfection, and additional types may be isolated in the future, as it isnot certain that all existing types have in fact been detected, and itis expected that mutation will result in appearance of previouslynon-existent forms. Of the five types now known, HPV-6 and HPV-11 areassociated with benign conditions, while HPV-16, HPV-18 and HPV-31appear to be associated with malignant transformation. All five of thesetypes, and presumably those still to be found, exhibit similarorganization of their DNA, so that all contain, for example, the openreading frames associated with the putative early (E) proteins and late(L) proteins. Depending on the use for which the peptide is intended,appropriate regions of the early or late putative proteins are selected.

The early proteins, as they are thought to be associated with vitalreplication and transformation, are particularly useful as portions ofimmunogens administered to production animals to generate antibodiesneeded for diagnosis of the progress of HPV infection. They are alsouseful as vaccine components. Reagents associated with the lateproteins, as these appear to be associated with capsid proteins, arealso useful as vaccines. They are also capable of raising antibodieswhich are diagnostic for the presence of free virus in the bloodstreamand are themselves useful in detecting antibodies raised against thewhole virus. Therefore, the first step in the analysis is to ascertainthe stage of vital infection which forms the subject matter for theutility.

The second decision point relates to whether the peptide is designed forapplications specific to a particular type or a broad spectrum ofgenital infection-causing HPV. If detection of antigen or antibodyassociated with a particular type is desired, it is desirable to have atype-specific peptide as the basis for the reagent or antiserum. On theother hand, if a vaccine protective against HPV genital infections ingeneral is desired, or if it is satisfactory to determine the presenceor absence of any HPV genital infection, then a peptide associated witha broad spectrum of these virions is desired.

To design species-specific vaccines, regions of the putative proteinsencoded by the open reading frames are selected which are not homologousfrom strain to strain, but characteristic of the particular type ofinterest. If a broad spectrum peptide is required, reagents of homologyare chosen. FIGS. 2-6 show comparisons of amino acid sequences forvarious open reading frames in several HPV types. These sequences showvarious regions of homology for these proteins: for example, in theearly protein encoding region E2, the region between positions 109-119is much more homologous than the region between 139 and 148 of thesequence; the region between residues 331 and 350 contains considerablehomology, whereas that between positions 241-260 does not.

The third level of selection relates to secondary structure andhydrophilicity. Regions are selected which are considered likely toraise antibodies in the host which are cross-reactive against the entireprotein on the basis of the ability of this particular subsection of theprotein to mimic its own secondary structure in the native state.Generally, if there is no additional information, one is limited to theuse of areas of conformational flexibility such as the N-terminal andC-terminal regions because these regions of the native protein areassumed to exhibit the same spectrum of conformations as the synthesizedpeptide. A difficulty with this approach, however, is that due to themultiplicity of possibilities, antibody production against a desiredconformation may represent a small portion of the antibodies raised andthe process is relatively inefficient.

A superior approach is based on ascertaining regions of the proteinwhich are likely to have the same conformation in the intact protein asis exhibited by the synthesized peptide. These are believed to beregions having reversed or beta turns as the formation of these turns isgenerally associated with the surface of the protein, and is alsodependent on the amino acid sequences in close proximity with eachother. Methods to ascertain the locations of the desired beta turns areknown in the art and described in the paper of Chou and Fassmanreferenced above. FIG. 7 shows an illustrative plot obtained accordingto these procedures showing the regions of alpha helixes, beta sheetsand reverse turns for the protein encoded by the E7 open reading frameof HPV-16. The results also provide data as to regions of hydrophobicityand hydrophilicity which are significant in the formation of these betaturns. Thus, using FIG. 7 as an illustration, those regions consideredlikely to exhibit the desired immunogenicity are illustrated as peptides1-5. In selecting these peptide regions, information is obtained fromthe plot which shows the presence of beta turns and associated regionsof hydrophilicity. This analysis can, of course, be done with respect tothe peptides encoded in the open reading frames of any of the desiredHPV types.

When the appropriate region is chosen by this analysis, peptides of 8-15amino acid residues which include this region are synthesized asdescribed below. It is believed that at least 8 amino acids are neededto provide sufficient sequence: peptides longer than 15 amino acidscould be synthesized, but at a sacrifice of economy.

Peptide Synthesis

As used herein, "peptide", "polypeptide", and "protein" are usedinterchangeably, and refer to amino acid sequences of a variety oflengths, either in their neutral (uncharged) forms or in forms which aresalts, and either free of modifications such as glycosylation, sidechain oxidation, or phosphorylation or containing these modifications.It is well understood in the art that amino acid sequences Containacidic and basic groups, and that the particular ionization stateexhibited by the peptide is dependent on the pH of the surroundingmedium when the protein is in solution, or that of the medium from whichit was obtained if the protein is in solid form. Also included in thedefinition are proteins modified by additional substituents attached tothe amino acid side chains, such as glycosyl units, lipids, or inorganicions such as phosphates, as well as modifications relating to chemicalconversions of the chains, such as oxidation of sulfhydryl groups. Thus,"peptide" or its equivalent terms is intended to include the appropriateamino acid sequence referenced, subject to those of the foregoingmodifications which do not destroy its immunospecific antigenicproperties.

All of the peptides of the invention are sufficiently short thatchemical synthesis, using methods now standard in the art, is feasible.A review of such methods is given by, for example, Margolin, A., et al,Ann Rev Biochem (1970) 39:841. In most of these procedures, theC-terminal amino acid is bound to a solid support, and reacted with thenext amino acid in sequence which has been protected at the amino groupto prevent self-condensation. After the initial coupling, the NH₂protecting group is removed, and the coupling process repeated with theamino acid next in order. Polypeptides of considerable chain length havebeen synthesized in this way. The only requirement is that the aminoacid sequence desired to be produced is known.

Recombinant DNA methodology provides an alternative way of synthesizingthe desired peptides. The DNA coding sequence for the desired peptide orprotein is ligated into an expression vector suitable for transforming arecipient cell, which is thus caused to express the gene and produce theprotein. The DNA coding sequences are sufficiently short to be preparedsynthetically using means known in the art: see. e.g., Edge, M. P., etal, Nature (1981) 292:756.

The coding sequence is placed under the control of control sequencescompatible with recombinant hosts in plasmids containing convenientrestriction sites for insertion of the desired coding sequence. Forexample, for bacterial hosts typical of such plasmids are pUC8, andpUC13 available from Messing, J., at the University of Minnesota: (see,e.g., Messing, et al, Nucleic Acids Res (1981) 9:309) or pBR322,available from New England Biolabs. Suitable promoters include, forexample, the β-lactamase (penicillinase) and lactose (lac) promotersystems (Chang. et al, Nature (1977) 198:1056 and the tryptophan (trp)promoter system (Goeddel, D., et al, Nucleic Acids Res (1980) 8:4057).The resulting bacterial expression vectors are transformed into suitablebacterial hosts using the calcium chloride method described by Cohen, S.N., et al, Proc Natl Acad Sci (USA) (1972) 69:2110, and thetransformants selected and cultured. Alternatively, these peptides canbe produced in nonbacterial recombinant hosts using appropriate controlsequences, vectors and transformation techniques.

Synthesis of Conjugates

Because the peptide sequences of the invention are considered too smallto be immunogenic, they are linked to carrier substances in order toconfer this property upon them. The carrier substances should beantigenically neutral in the subject generating the antisera.

By "substantially antigenically neutral carrier" is meant a material towhich the peptides of the invention may be attached to render themimmunogenic, but which does not itself elicit antibodies which will bedetrimental to the host, or contain antigenic sites which interfere withthe antigenic function of the invention peptides. For example, insubjects which are not beef eaters, such as rabbits or mice, bovineserum albumin (BSA) could be used. For human use, however, carriers arelimited to proteins which do not raise antibodies to materials commonlyand nonpathogenically encountered by humans. For example, human serumalbumin (HSA) or tetanus toxoid protein could be used.

The conjugates can be formed in a variety of ways. For example, thereare a large number of heterobifunctional agents which generate adisulfide link at one functional end group and a peptide link at theother, and these have been used extensively. The most popular of theseis N-succidimidyl-3-(2-pyridyl dithio) propionate (SPDP). This reagentcreates a disulfide linkage between itself and a cysteine residue in oneprotein and an amide linkage through the amino on a lysine, or otherfree amino group in the other. A variety of such disulfide/amide formingagents are known. See, for example, Immun Rev (1982) 62:185. Otherbifunctional coupling agents form a thioether rather than a disulfidelinkage. Many of these thioether forming agents are commerciallyavailable and include reactive esters of 6-maleimidocaproic acid, 2bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid and the like. The carboxyl groups can beactivated by combining them with succinimide or1-hydroxy-2-nitro-4-sulfonic acid, sodium salt. A particularly preferredcoupling agent for the method of this invention is succinimidyl4-(N-maleimido-methyl) cyclohexane-1-carboxylate (SMCC) obtained fromPierce Company, Rockford, Ill.

The foregoing list is not meant to be exhaustive, and modifications ofthe named compounds can clearly be used. However, if a disulfide orthioether linkage to the peptide is to be employed, and the peptidecontains no convenient cysteine, an additional cysteine residue ateither terminus can be added when the peptide is prepared. As onlyshorter peptides require conjugation to carrier, these residues can beincluded conveniently during chemical synthesis.

Vaccine Preparation

Preparation of vaccines which contain peptide sequences as activeingredients is well understood in the art. Typically, such vaccines areprepared as injectables, either as liquid solutions or suspension: solidforms suitable for solution or suspension in liquid prior to injectionmay also be prepared. The preparation may also be emulsified. The activeimmunogenic ingredient is often mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof. In addition, if desired,the vaccine may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, or adjuvants whichenhance the effectiveness of the vaccine. The vaccines areconventionally administered parenterally, by injection, for example,either subcutaneously or intramuscularly. Additional formulations whichare suitable for other modes of administration include suppositoriesand, in some cases, oral formulations.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, capacity of the subject's immune system to synthesizeantibodies, and the degree of protection desired. Precise amounts ofactive ingredient required to be administered depend on the judgement ofthe practitioner and are peculiar to each individual. However, suitabledosage ranges for subcutaneous or muscular injection are of the order to50-500 μg active ingredient per individual. Suitable regimes for initialadministration and booster shots are also variable, but are typified byan initial administration followed in one-two week intervals by asubsequent injection or other administration.

Preparation of Antisera

For preparation of diagnostic antisera, hyperimmune sera can beprepared, for example, in rabbits using standard immunization techniquescomprising an initial injection and boosting. The sera can beperiodically titrated in a solid phase assay against the immunogenicpeptide. For preparation of monoclonal panels, the immunized animals aresacrificed and spleen cells harvested for immortalization to obtaincells capable of producing antibodies. The supernatants of successfulimmortalized cells are screened for the production of antibodiesreactive with the injected peptide. If therapeutic uses are to be madeof the monoclonals, the preparation of antisera and immortalization mayinvolve techniques capable of conferring suitable speciescharacteristics on the secreted antibodies. Techniques to obtainantibodies with human characteristics are summarized and disclosed inTeng, N. N. H., at al. in Human Hybridomas and Monoclonal Antibodies(1985) Engleman, E., et al. Ed., Plenum Press, p. 71.

Diagnostics

The prepared antisera can be used in a variety of diagnostic assays. Allof these are, of course, immunoassays and the design of such assays isof tremendous variety. Perhaps most convenient are solid phase supportedimmunoassays which employ "sandwiches" in which at least one layer iscomprised of the antibodies of the invention and another layer iscomprised of sample. In one possible embodiment, the antisera preparedaccording to the method of the invention are to coat a solid support,such as, for example, a microtiter plate to provide a surfacespecifically reactive with the HPV components of the sample to betested. The plates are then treated with sample, and then with anadditional antibody also reactive with the desired HPV antigen. Thethird layer may itself contain a radioactive, fluorescent, or enzymiclabel or may be reactive with still another layer containing suchlabels. The variety of protocols for such assays is well understood inthe art, and any suitable protocol is acceptable.

With regard to the indications for which the antibodies of the inventionare useful in diagnosis, the most commonly encountered use is clearlythe diagnosis of the presence or absence of HPV-16 or other relevant HPVeither in a pap smear or in cervical biopsy or in biopsies of othertissue. The presence of HPV-16, specifically, and also of HPV-18 orHPV-31, in a cervical biopsy or pap smear is indicative of a high riskfor malignant transformation resulting in cervical carcinoma.

The progress of HPV infection to result in the malignant state isbelieved to pass through three CIN stages, commonly labeled CIN1, CIN2,and CIN3 before finally converting to a malignant transformed carcinoma.The various stages may be associated with expression of different genesof the HPV virus with resulting changes in the level of differentiationof the infected tissue. Therefore, an objective distinction between CIN3and CIN2 can be made using these antisera, since the differentiatedstate associated with CIN3 fails to produce a positive response againstthe antisera raised against the peptides 1-4 of the invention, whilethat associated with CIN2 does do so. Thus, if the morphologicalcharacteristics as to the stage of development of the lesion areunclear, these two stages can be distinguished by virtue of theirreactivity with the antisera.

In addition, the antisera are useful in detecting the HPV virus in bloodor serum, thus providing a basis for antigen detection in these fluids,or in foreign tissues which would indicate the presence of metastasesfrom an original site. The results of treatment of carcinoma associatedwith HPV infection can also be followed by assessing the levels ofspecies reactive with these antisera in serum or other tissues. Thus, ifthe disease is treated, for example, by administration of interferon,the effect of this drug can be monitored by tracking the levels of HPVantigens in serum using the antibodies in this assay.

Conversely, the peptides of the invention can be used in immunoassays totest for the presence or absence of anti-HPV antibodies in serum. Theprotocol for such assays is similar to that associated with the use ofantisera to test for the presence or absence of antigen, except that thesample and reagent are interchanged. These proteins are a convenientsource of pure HPV material which thereby permit assays with a minimumof cross reaction.

Therapeutic Uses

The antibodies can also be used therapeutically so long as they arecompatible with the host to be treated. Monoclonal preparations havingthe proper species characteristics are most suitable for thisapplication of the invention which is conducted by injecting into aperson already suffering from HPV infection sufficient antibodypreparation to combat the progress of the infection.

As noted above, the peptides themselves, in addition to being used asvaccines in advance of infection for protection against the disease, canalso be used as an immunomodulation agent when the subject is alreadyinfected. By supplying additional immunogenic peptides, the antibodyresponse mounted by the patient is fortified, and is thus more effectivein disease control.

Kits

The diagnostic antibodies or peptides of the invention can convenientlybe packaged into kits, for the conduct of the immunoassays in which theyare the essential reagent. The components of such kits include,typically, the peptide or antibody preparation of the invention, labeledantibody or other protein to permit detection, and optionally, suitablesupports and containers for conducting the protocols described. Inaddition, such kits will contain instructions for utilizing the peptidesor antisera of the invention in the context of the samples to be testedand the equipment and reagents provided.

EXAMPLES

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLE 1 Preparation of Peptides

Peptides 1-17, further containing a C-terminal cysteine residue areprepared by the solid phase techniques disclosed in Chirgwin, J. M., etal, Biochem (1979) 18:5294 using t-Boc protected amino acids and aminoacid derivatized polystyrene resin, supplied by Peninsula LaboratoriesInc., Belmont, Calif. Asp, Glu, Thr, and Set side chains are protectedas O-benzyl esters; Arg and His side chains are protected with tosylgroups; Cys is protected with p-methoxybenzyl; Lys byO-chlorobenzyloxycarbonyl, and Tyr by 2,6-dichloro benzyl. The couplingsare performed with 2.5-3 molar excess of t-Boc amino acid anddicyclohexylcarbodiamide (DCC). For couplings of Asn or Gln, a 2.5-foldmolar excess of N-hydroxytriazole is also included. Coupling ismonitored with ninhydrin and coupling is continued until 99% efficiencyis obtained.

After synthesis of the desired chain, protecting groups and resin arecleaved simultaneously by anhydrous hydrogen fluoride in the presence ofdimethyl sulfoxide and anisol. The cleaved peptide is extracted withether, isolated from the resin by extraction with 5% acetic acid, andlyophilized several times. The purity of the final product is determinedby reverse phase HPLC on Licosorb RP18 (Merck Dormstat FRG) and by aminoacid analysis.

EXAMPLE 2 Conjugation to Carrier Protein

Each of the peptides 1-17, synthesized in Example 1 is conjugated tothyroglobin or to bovine serum albumin. In either case. 10 mg of thecarrier protein are dissolved in 3 ml PBS. pH 7.4. and mixed with 1 mldistilled DMF containing 5 mg of the cross-linkerm-malenimidobenzoyl-N-hydroxysuccinimidyl ester (MBS) for thyroglobinand succinimidyl 4-(N-malenimido methyl)cyclohexane-1-carboylate (SMCC)for bovine serum albumin.

In the meantime, each of the peptides 1-17 is reduced with sodiumborohydride for 15 min on ice to ensure the presence of a freesulfhydryl. After destroying excess borohydride with HCl, theneutralized and reduced peptide is combined with the carriercross-linker conjugate and stirred overnight at room temperature. Theresulting peptide carrier conjugate is isolated by gel filtration onSephadex G25 in 0.1M ammonium bicarbonate buffer, pH 7.5. and the molarratio of peptides to carrier determined in the product.

EXAMPLE 3 Preparation of Antisera

Hyperimmune sera are prepared in female New Zealand white rabbits usingthe conjugates prepared in Example 2. Two rabbits are immunized for eachof the peptides 1-17 carrier conjugates. Five hundred μg ofthyroglobin-MBS-peptide conjugate are emulsified in Freund's adjuvantand injected subcutaneously and intramuscularly at multiple cites. Therabbits are boosted at six weeks with the same immunogen emulsified withincomplete Freund's adjuvant, and one week later the animals are bled bycardiac puncture. The animals are boosted one week later, bled two weekslater, boosted a third time three weeks later and bled a third time fourweeks after the initial cardiac puncture. The antisera are thenevaluated in the microtiter solid phase binding assay usingpeptide-SMCC-BSA conjugate as antigen.

EXAMPLE 4 Titration of Antisera

Wells of disposable polystyrene U microtiter plates are coated withpeptide-BSA conjugate in 0.1M sodium carbonate buffer, pH 9.6 for 12 hrat room temperature. The wells are then washed three times withNaCl-brij. Serially diluted antisera are added in PBS-brij-BSA andincubated at 37° C. for 2 hr and washed. Approximately 20,000 cpm of ¹²⁵I-protein A in PBS-BSA-brig is added to each well and incubated at 37°C. for 1 hr and washed. The wells are assayed for radioactivity in agamma scintillation counter, negative controls are nonsensitized wells,wells which are not exposed to antisera and wells exposed to preimmunesera. Each sample is performed in triplicate and recorded as the mean.

EXAMPLE 5 Use of Antisera in Immunoassays

Four of the antisera prepared in Example 4 are used to assess cervicalswabs and biopsies for the presence of HPV virus in correlation tovarious CIN stages and malignant cervical carcinoma. Samples areobtained from patients referred to dysplasia clinics for history ofabnormal pap smears. Cervical biopsy material is frozen and stored.

To conduct the assays, several protocols may be employed, includingimmunoperoxidase staining of pap smears and cervical biopsies, dotimmunobinding of cervical smears, Western blot, and immunoprecipitation.

For cervical swabs immunobinding on nitrocellulose paper is convenientlyemployed. Cervical swabs are spotted onto a 1 cm² section ofnitrocellulose paper, pore size 0.2 μm as described by Jahn, et al, ProcNatl Acad Sci (USA) (1984) 81:1684-1687. The paper is air dried andfixed for 15 min in a 10% v/v acetic acid, 25% v/v isoproponyl solution,rinsed, preincubated for 5 min in Tris-buffered saline and incubated inblocking buffer consisting of Tris-saline-5% BSA. The paper is thenincubated with antiserum to peptides 1, 2, 3 and 4 above inTris-saline-BSA-0.1% Triton X-100 for 2 hr, rinsed and immersed again inblocking buffer for 30 min. The paper is then incubated in 300,000 cpmof ¹²⁵ I-protein A in Tris-saline-BSA-Triton X-100 for 1 hr at 37° C.,washed and dried. The squares are cut out and assayed for radioactivityin a gamma scintillation counter.

For biopsies, immunoperoxidase staining is most convenient. Theavidin/biotin method described by Hsu, S. M., et al. Advances inImmunohistochemistry (1984) Medical Publishers, pp. 31-42 was used.

The tissue sections are immersed in 3% hydrogen peroxide to blockendogenous peroxidase activity. The slides are then washed in distilledwater and PBS, and then immersed in 3% normal goat serum.(VectorLaboratories) to block nonspecific binding. The peptide antisera arethen added in dilutions ranging from 1:100 to 1:2000 and incubatedovernight at 4° C. The slides are rinsed in PBS and incubated with goatanti-rabbit IgG, and then reacted with avidin/biotin complex (ABC)reagent (Vector Laboratories) for a minimum of 30 min. Labelingantibodies (ABC stain) is added for 30 min and the slides again rinsedin PBS. Chromogen reaction using diaminobenzedine (DAB) solution (0.05%Tris pH 7.6, DAB, 30% hydrogen peroxide) is then developed for 5 min,and the samples are rinsed, counterstained with hematoxylin and coverslipped. A positive staining is indicated by a black-brown chromogenicprecipitate of DAB.

FIG. 8 shows the results of staining of a single biopsy which isassessed as CIN 2 by pathology. FIG. 8A shows this section stained usingrabbit antiserum prepared against peptide 1, but preadsorbed with thepeptide by incubating the serum in the presence of excess peptide 1. AsFIG. 8A shows, no DAB precipitate is detectable: the epithelial layer isclearly distinguishable from the stroma by apparent cell morphology withclear definition of the basement membrane. FIG. 8B is the same sectionstained using the same antisera, but unadsorbed with peptide 1. Theepithelium clearly shows the presence of the CIN2 characteristics inthis tissue. The HPV antigen is associated with this stage asdemonstrated by the brown stain apparent in the photograph. The antigendoes not appear in the stroma, since penetration of the basementmembrane has not yet occurred in CIN2. FIG. 8C is a magnification of thestained area at the surface of the epithelium shown in FIG. 8B.

FIG. 9 shows the ability of the immunoperoxidase method of the inventionto detect the presence of a single malignant cell in the stroma. FIG. 9is a magnified view of stained tissue from a patient where malignanttransformation has already occurred and a pathologic diagnosis ofcervical carcinoma has already been made. The cell at the center of thephotograph is an isolated malignant cell exhibiting the HPV antigens.

The results illustrated in FIGS. 8 and 9 were obtained in the course ofa study involving eleven patients as described above. In these assays,controls were run to show that no immunoreaction (brown stain) was everobserved using pre-immune serum or serum previously incubated with theimmunizing peptide. These controls were run in all tests. Additionalcontrols were also run with antisera to peptide 1, using samples knownto contain HPV-16, HPV-18, HPV-6 or HPV-11. Only HPV-16 and 18 gavepositive results and higher color intensity was obtained with HPV-16;this cross reactivity with HPV-18 shows that peptide 1 generatescross-type reacting antisera. Normal cervical swabs were also used ascontrols and no reaction was obtained using antisera obtained from anyof the four peptides tested.

However, patients having known dysplasias did show positive results asset forth in Table 1 below. The results shown in Table 1 indicate thatthe test detects presence of the virus and tracks the progress of tissuedifferentiation caused by the stepwise expression of the vital genome.

                  TABLE 1                                                         ______________________________________                                                            Peptide #                                                                     1    2      3      4                                      ______________________________________                                        2 patients CIN1                                                                           HPV-16 DNA(-) -      -    -    -                                  6 patients CIN1                                                                           HPV-16 DNA(+) +      +    +    +                                  4 patients CIN2                                                                           HPV-16 DNA(+) +      +    +    +                                  2 patients CIN3                                                                           HPV-16 DNA(+) -      -    -    -                                  1 patient SSC                                                                             HPV-16 DNA(+) +      -    +    +                                  normal Adj CIN3                                                                           HPV-16 DNA(+) +      +    +    +                                  ______________________________________                                    

The first two patients, listed in row 1 of Table 1, were assessed ashaving CIN1 stage lesions by pathology. However, the absence of HPV-16virus in these patients (indicated by the (-)) was determined bySouthern blot DNA hybridization performed on the smear. As shown, thesepatients also tested negative for the presence of HPV-16 using theantisera. On the other hand, the six patients also showing CIN1 bypathology, but shown to harbor HPV-16 by Southern blot DNA hybridization(labeled (+)), gave positive results with the antisera. The fourpatients assessed as CIN2 gave clear positive tests with the antiserafor the presence of HPV.

However, two patients assessed as CIN3 were unreactive with all serapresumably because of the differentiation state of the tissue at thisstage. The biopsy from one patient was assessed independently, but theother CIN3 tissue was obtained adjacent to one of the CIN2 samples inthe preceding row. This confirms that the test with antiserum makespossible an objective differentiation between CIN2 and CIN3. This issometimes difficult to ascertain by visual inspection; i.e. the findingsof a single pathologist will generally be internally consistent, butthere are frequently differences between the assessments given bydifferent pathologists.

As shown in the next-to-bottom line of the table, a patient having aknown carcinoma was positive with respect to peptides 1, 3 and 4 butnegative with respect to peptide 2, presumably indicating alteration ingenomic integration, transcription, or post-transcription events whichmay occur when the cells become malignant. The last line of the tablegives the results for normal tissue obtained from a patient who also hadCIN3 tissue which had tested negative against all antisera, but whichCIN3 tissue had been shown to contain HPV-16 DNA by Southern blot. Thepositive results indicate that latent virus, and even gene expressionproducts remain in the normal cells when adjacent tissues are in laterstages of the infection.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 17                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       SerArgSerSerA rgThrArgArgGluThrGlnLeu                                         1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       PheG lnAspProGlnGluArgProArgLysLeuProGlnLeuCys                                151015                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ThrGluLeuGlnThrThrIleHisAspIleIleLeuGluCys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       LeuArgArgGluValTyrAspPheAlaPheArgAspLeuCys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AspLysLysGlnArgPheHisAsnIleArg                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GlyProAlaGlyGlnAlaGluProAspArgAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 9 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AspThrProThrLeuHisGluTyrMet                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                     (B) TYPE: amino acid                                                         (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AsnAspSerSerGluGluGluAspGluIleAspGly                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 ( A) LENGTH: 11 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       LeuGlnLeuThrLeuGluThrIleTyrAsnSer                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      IleIleArgGlnHisLeuAlaAsnHisPro                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 13 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      HisProAlaAlaThrHisThrLysAlaValAlaLeuGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      SerGluTrpGlnArgAspGlnPheLeuSerGlnVal                                          15 10                                                                         (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      AspGlnAspGlnSerGlnThrProGluThrPro                                             15 10                                                                         (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GlySerThrTrpProThrThrProProArgProIleProLysPro                                 1 51015                                                                       (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ArgLeuTyrLeuHisGluAs pGluAspLysGluAsn                                         1510                                                                          (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AlaProIleLe uThrAlaPheAsnSerSerHisLysGlyCys                                   1510                                                                          (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                       GluSerAlaAsnAlaSerThrSerAlaThrThrIleCys                                      1510                                                                      

We claim:
 1. A method for indicating the presence of human papillomavirus in a clinical sample which comprises the steps of:(a) providing anantibody-containing composition, said composition containing antibodyimmunospecific for an epitope having an amino acid sequence selectedfrom the group consisting of(1)Ser-Arg-Ser-Ser-Arg-Thr-Arg-Arg-Glu-Thr-Gln-Leu (SEQ ID NO:1)(representing residues 147-158 of E6 except Ser was substituted for Cysat position 1); (2)Phe-Gln-Asp-Pro-Gln-Glu-Arg-Pro-Arg-Lys-Leu-Pro-Gln-Leu-Cys (SEQ IDNO:2), representing residues 9-23 of E6; (3)Thr-Glu-Leu-Gln-Thr-Thr-Ile-His-Asp-Ile-Ile-Leu-Glu-Cys (SEQ ID NO:3),representing residues 24-37 of E6; (4)Leu-Arg-Arg-Glu-Val-Tyr-Asp-Phe-Ala-Phe-Arg-Asp-Leu-Cys (SEQ ID NO:4),representing residues 45-58 of E6; (5)Asp-Lys-Lys-Gln-Arg-Phe-His-Asn-Ile-Arg (SEQ ID NO:5), representingresidues 127-136; (6) Gly-Pro-Ala-Gly-Gln-Ala-Glu-Pro-Asp-Arg-Ala (SEQID NO:6), representing residues 40-50 of E7; (7)Asp-Thr-Pro-Thr-Leu-His-Glu-Tyr-Met (SEQ ID NO:7), representing residues4-12 of E7; (8) Asn-Asp-Ser-Ser-Glu-Glu-Glu-Asp-Glu-Ile-Asp-Gly (SEQ IDNO:8), representing residues 29-40 of E7; (9)Leu-Gln-Leu-Thr-Leu-Glu-Thr-Ile-Tyr-Asn-Ser (SEQ ID NO:9), representingresidues 75-85 of E2; (10) Ile-Ile-Arg-Gln-His-Leu-Ala-Asn-His-Pro (SEQID NO:10), representing residues 210-219 of E2; (11)His-Pro-Ala-Ala-Thr-His-Thr-Lys-Ala-Val-Ala-Leu-Gly (SEQ ID NO:11),representing residues 218-230 of E2; (12)Ser-Glu-Trp-Gln-Arg-Asp-Gln-Phe-Leu-Ser-Gln-Val (SEQ ID NO:12),representing residues 339-350 of E2; (13)Asp-Gln-Asp-Gln-Ser-Gln-Thr-Pro-Glu-Thr-Pro (SEQ ID NO:13), representingresidues 48-58 of E4; (14)Gly-Ser-Thr-Trp-Pro-Thr-Thr-Pro-Pro-Arg-Pro-Ile-Pro-Lys-Pro (SEQ IDNO:14), representing amino acids 20-34 of E4; (15)Arg-Leu-Tyr-Leu-His-Glu-Asp-Glu-Asp-Lys-Glu-Asn (SEQ ID NO:15),representing amino acids 476-487 of E1; and (16)Ala-Pro-Ile-Leu-Thr-Ala-Phe-Asn-Ser-Ser-His-Lys-Gly-Cys (SEQ ID NO:16),representing amino acids 218-230 of E2; wherein all the foregoing arederived from Type 16; and (17)Glu-Ser-Ala-Asn-Ala-Ser-Thr-Ser-Ala-Thr-Thr-Ile-Cys (SEQ ID NO:17),representing amino acids 6-17 of the E6 reading frame of Type 6B, (b)contacting said clinical sample with said composition underantibody-antigen complex formation conditions, (c) detecting theformation of antibody-antigen complex resulting from the contacting ofstep (b), and (d) relating the detection of complex of step (c) to thepresence in the clinical sample of human papilloma virus.
 2. A methodfor distinguishing CIN2 and CIN3 stages of human papilloma virusinfection in a clinical sample selected from the group consisting of acervical biopsy and a cervical smear which comprises the steps of:(a)providing an antibody-containing composition, said compositioncontaining antibody immunospecific for an epitope having an amino acidsequence selected from the group consisting of(1)Ser-Arg-Ser-Ser-Arg-Thr-Arg-Arg-Glu-Thr-Gln-Leu (SEQ ID NO:l)(representing residues 147-158 of E6 except Ser was substituted for Cysat position 1); (2)Phe-Gln-Asp-Pro-Gln-Glu-Arg-Pro-Arg-Lys-Leu-Pro-Gln-Leu-Cys (SEQ IDNO:2), representing residues 9-23 of E6; (3)Thr-Glu-Leu-Gln-Thr-Thr-Ile-His-Asp-Ile-Ile-Leu-Glu-Cys (SEQ ID NO:3),representing residues 24-37 of E6; (4)Leu-Arg-Arg-Glu-Val-Tyr-Asp-Phe-Ala-Phe-Arg-Asp-Leu-Cys (SEQ ID NO:4),representing residues 45-58 of E6; (5)Asp-Lys-Lys-Gln-Arg-Phe-His-Asn-Ile-Arg (SEQ ID NO:5), representingresidues 127-136; (6) Gly-Pro-Ala-Gly-Gln-Ala-Glu-Pro-Asp-Arg-Ala (SEQID NO:6), representing residues 40-50 of E7; (7)Asp-Thr-Pro-Thr-Leu-His-Glu-Tyr-Met (SEQ ID NO:7), representing residues4-12 of E7; (8) Asn-Asp-Ser-Ser-Glu-Glu-Glu-Asp-Glu-Ile Asp-Gly (SEQ IDNO:8), representing residues 29-40 of E7; (9)Leu-Gln-Leu-Thr-Leu-Glu-Thr-Ile-Tyr-Asn-Ser (SEQ ID NO:9), representingresidues 75-85 of E2; (10) Ile-Ile-Arg-Gln-His-Leu-Ala-Asn-His-Pro (SEQID NO:10), representing residues 210-219 of E2; (11)His-Pro-Ala-Ala-Thr-His-Thr-Lys-Ala-Val-Ala-Leu-Gly (SEQ ID NO:11),representing residues 218-230 of E2; (12)Ser-Glu-Trp-Gln-Arg-Asp-Gln-Phe-Leu-Ser-Gln-Val (SEQ ID NO:12),representing residues 339-350 of E2; (13)Asp-Gln-Asp-Gln-Ser-Gln-Thr-Pro-Glu-Thr-Pro (SEQ ID NO:13), representingresidues 48-58 of E4; (14)Gly-Ser-Thr-Trp-Pro-Thr-Thr-Pro-Pro-Arg-Pro-Ile-Pro-Lys-Pro (SEQ IDNO:14), representing amino acids 20-34 of E4; (15)Arg-Leu-Tyr-Leu-His-Glu-Asp-Glu-Asp-Lys-Glu-Asn (SEQ ID NO:15),representing amino acids 476-487 of E1; and (16)Ala-Pro-Ile-Leu-Thr-Ala-Phe-Asn-Ser-Ser-His-Lys-Gly-Cys (SEQ ID NO:16),representing amino acids 218-230 of E2; wherein all the foregoing arederived from Type 16; and (17)Glu-Ser-Ala-Asn-Ala-Ser-Thr-Ser-Ala-Thr-Thr-Ile-Cys (SEQ ID NO:17),representing amino acids 6-17 of the E6 reading frame of Type 6B, (b)contacting said clinical sample with said composition underantibody-antigen complex formation conditions, (c) detecting theformation of antibody-antigen complex resulting from the contacting ofstep (b), and (d) relating the detection of complex of step (c) to thepresence in the clinical sample of CIN3 stage human papilloma virusinfection.